The overall goal of this project is to develop an attenuated rhesus cytomegalovirus-based vaccine against simian immunodeficiency virus (RhCMV/SIV) that is unable to replicate in cells and tissues associated with CMV transmission and disease. We have shown that live RhCMV/SIV is an effective vaccine that induces SIV protective immunity in rhesus macaques. In order to translate our findings into a human CMV (HCMV)/HIV vaccine that would be safe for all potential patients including immunocompromised individuals, the CMV vaccine vector needs to be attenuated without losing the ability to induce protective immunity. CMV can replicate in a wide variety of cells and tissues in the host, including: epithelial cells in glandular tissue (salivary glands and breast), lung, kidney, as well as hepatocytes in the liver and neurons in the central nervous system (CNS). Myeloid and endothelial cells are also considered persistent sites for CMV in the host. The overall goal of Project 2 is to modulate the ability of CMV to replicate in these critical cell types in order to increase safety without compromising vaccine efficacy. We hypothesize that elimination of all the epithelial cell tropic genes will reduce pathogenicity and eliminate viral shedding into saliva and urine. Therefore, in the first specific aim of this project, we will delete RhCMV epithelial cell tropism genes to further abrogate the ability of the virus to replicate in this cell type. As an additional method to attenuate the RhCMV/SIV vaccine, we will use a novel approach to inactivate virus in tissues associated with CMV disease and dissemination by using a microRNA-based strategy to specifically inactivate essential CMV genes during viral replication in CNS, liver, and myeloid cells. These RhCMV/SIV tropism deficient viruses will be analyzed for SIV immunogenicity and attenuation in rhesus macaques in Specific Aim 2. In the last specific aim, we will translate these findings into a human CMV/HIV vector that will be tested in a newly established humanized mouse model. These studies will result in the design of an HCMV/HIV vaccine that will be ready for clinical studies.